CN101816715A - Method for measuring content of Danshensu in shenshuaining capsules by utilizing HPLC method - Google Patents
Method for measuring content of Danshensu in shenshuaining capsules by utilizing HPLC method Download PDFInfo
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- CN101816715A CN101816715A CN200910218240A CN200910218240A CN101816715A CN 101816715 A CN101816715 A CN 101816715A CN 200910218240 A CN200910218240 A CN 200910218240A CN 200910218240 A CN200910218240 A CN 200910218240A CN 101816715 A CN101816715 A CN 101816715A
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- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 title claims abstract description 51
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- 238000012360 testing method Methods 0.000 description 12
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Abstract
The invention provides a method for measuring the content of Danshensu in shenshuaining capsules by utilizing an HPLC method, comprising the following steps: 1, weighing shenshuaining capsules into a measuring flask, crushing, adding methanol, performing ultrasound in water bath, placing in the ice-water bath, taking out, adding methanol, centrifuging, and taking the supernate as the test solution of the shenshuaining capsules; 2, weighing Danshensu sodium comparison products, placing in the measuring flask, performing ultrasound in water bath, taking out, placing in the ice-water bath, taking out, adding methanol to shake up for being served as the comparison products solution; 3, leading the chromatographic column to be Inertsil ODS-3 (4.6X 150mm 5mu m), the mobile phase to be methanol-water-glacial acetic acid, the volume ratio to be 19:80:1, the flow velocity to be 1.0ml/min, the detected wavelength to be 281nm, the sensitivity to be 0.01AUFS, the sample size to be 10 mu L, the column temperature to be 40 DEG C, and the theoretic plate number to be no less than 5000 calculated by chromatographic peak of the Danshensu; and 4, injecting Danshensu sodium comparison products solution and shenshuaining capsules test solution into liquid chromatograph, and measuring the chromatographic peak presented by shenshuaining capsules test solution with the same chromatographic retention as the Danshensu sodium comparison products solution. The invention provides a new comparison standard for accurately assessing the quality of shenshuaining capsules, thus strengthening controllability of product quality of shenshuaining capsules and being beneficial for comprehensive monitoring of product quality of shenshuaining capsules.
Description
Technical field
The present invention relates to a kind of HPLC of use method and measure the method for Chinese patent medicine content, concrete is the method that a kind of HPLC of use method is measured content of Danshensu in the SHENSHUAINING capsule.
Background technology
The compound Chinese medicinal preparation that the SHENSHUAINING capsule is made up of Radix Salviae Miltiorrhizae, Radix Et Rhizoma Rhei, Radix Pseudostellariae, Radix Achyranthis Bidentatae, Rhizoma Coptidis, Poria, Flos Carthami etc., wherein Radix Salviae Miltiorrhizae is a monarch drug.Radix Salviae Miltiorrhizae is the dried roots of Labiatae clary; the modern Chinese medicine pharmaceutical research shows; when Radix Salviae Miltiorrhizae can be protected original position kidney-heat ischemia effectively to infringement that renal tubular epithelial caused; to kidney-heat ischemia bleeding from anus creatine concentration, creatinine clearance rate and free water clearance adjusted effectively certain protection and repair can, can increase the rabbit kidney blood flow volume.Can reduce blood urea nitrogen, creatinine, glomerular filtration rate (GFR), renal plasma flow (RPF), renal blood flow (RBF) are significantly increased, renal function obviously improves, and can significantly increase the discharge of carbamide in the urine, flesh tincture, sodium and Phos.Can reduce serum creatinine and urine N-acetyl-β-D-glucosamine glycoside enzyme (NAG enzyme).Its mechanism and stable lysosome membrane stop stream in the metabolic defect in cellular calcium ion, and anticoagulation, antithrombotic formation, antiplatelet aggregation, blood vessel dilating, increase be huge, and to bite phagocytic function etc. relevant.Danshensu wherein may be one of main effective ingredient of Treated with Radix Salviae Miltiorrhizae nephropathy aspect.
Along with to Chinese medicine research further deeply, people recognize gradually to Chinese medicine preparation, simply can not appropriately reflect the inherent quality of Chinese medicine with the quality control pattern of doctor trained in Western medicine synthetic drug.HPLC is meant Chinese medicine fingerprint, Chinese medicine fingerprint has been used the notion of prudence fingerprint identification, and the utilization modern analytical technique obtains spectrum or stratographic collection of illustrative plates common, that have the common composition of distinctive composition in certain Chinese crude drug, extract or the Chinese patent medicine.Chinese medicine fingerprint is made up of the characteristic signal of measured object, has individual uniqueness, has represented the feature of measured object.Chinese medicine fingerprint is significant for the quality of effective control Chinese crude drug, extract or Chinese patent medicine, and deeper quality evaluation pattern can be provided.
Summary of the invention:
The objective of the invention is to, a kind of new solution of using the HPLC method to measure the method for content of Danshensu in the SHENSHUAINING capsule is proposed, technical scheme of the present invention has fast, accurately measures the content of danshensu in the SHENSHUAINING capsule, effectively controls the characteristics of SHENSHUAINING capsule product quality.
Technical scheme of the present invention: use the HPLC method to measure the method for content of Danshensu in the SHENSHUAINING capsule, step is as follows:
(1) SHENSHUAINING capsule test solution preparation: accurately take by weighing 12 of SHENSHUAINING capsules (0.35g/ grain) in the 25mL measuring bottle, crush gently, add methanol 15mL, ultrasonic 10min in 40 ℃ of water-baths, put in the ice-water bath 5 minutes, and took out, add methanol to scale, centrifugal 5min gets supernatant and is the Yiganning capsule test solution that declines;
(2) preparation of reference substance solution: precision takes by weighing danshensu sodium reference substance 1.66mg, puts in the 10mL measuring bottle, and ultrasonic 10min in 40 ℃ of water-baths takes out, and puts in the ice-water bath 5 minutes, takes out, and adds methanol to scale and shakes up, in contrast product solution;
(3) chromatographic condition: chromatographic column is Inertsil ODS-3 (4.6X150mm 5 μ m); Mobile phase is methanol-water-glacial acetic acid, and volume ratio is 19: 80: 1; Flow velocity: 1.0ml/min; The detection wavelength is 281nm; Sensitivity: 0.01AUFS; Sample size: 10 μ L; Column temperature: 40 ℃, theoretical cam curve: be not less than 5000 by the danshensu chromatographic peak;
(4) algoscopy: accurate respectively danshensu sodium reference substance solution and each 10mL of SHENSHUAINING capsule test solution of drawing, inject chromatograph of liquid, measure SHENSHUAINING capsule test solution and should present and the identical chromatographic peak of danshensu sodium reference substance solution chromatographic retention.
Existing preparation before the described danshensu sodium reference substance of step (2) uses, lucifuge, cryopreservation.The methanol that is adopted is chromatographically pure, and water is pure water, and other reagent is analytical pure.
The various raw material of Chinese medicine that Chinese medicine preparation adopted are because of reasons such as the place of production, weather, seasons, and wherein contained active ingredient is not quite similar.The raw material and identical preparation technology of equal number often appear adopting in the production, in the product of producing active ingredient inequality, make that the controllability of Chinese medicine preparation quality is bad.The present invention sets up macroscopical finger printing to the effective constituent danshensu chemical constituent that mainly contains in the SHENSHUAINING capsule product with high performance liquid chromatography, the reuse danshensu sodium is that reference substance is set up the danshensu sodium finger printing, compare with the danshensu sodium finger printing partly in the SHENSHUAINING capsule fingerprint pattern with the danshensu sodium finger printing, thereby accurately measure the content of danshensu in the SHENSHUAINING capsule, effectively control SHENSHUAINING capsule product quality.This collection of illustrative plates is being represented the main pharmacologically active of SHENSHUAINING capsule, can characterize capsular quality of SHENSHUAINING and effect effectively.The present invention provides new reference standard for the decline quality of Yiganning capsule of accurate evaluation of renal.Compare with conventional quality standard, the present invention is deeper quality evaluation pattern, strengthened the controllability of SHENSHUAINING capsule product quality, can realize the chemical analysis of SHENSHUAINING capsule maximum possible is detected, helped SHENSHUAINING capsule product quality overall monitor.The present invention adopts scientific method, by the resulting practicable method of a large amount of experiments, has good repeatability and stability.Have aborning easy to use, characteristics fast and accurately.
Description of drawings
Fig. 1 is reference substance, need testing solution by 2.2,2.3 method preparations, and under 2.1 chromatographic condition, sample detection obtains each batch SHENSHUAINING capsule high performance liquid chromatography stacking chart successively.Among the figure: R is a reference substance Chinese collection of illustrative plates; S1-S13 is the SHENSHUAINING capsule sample, and 8 is danshensu, and 9 is Jian.
Fig. 2 changes the chromatogram of glacial acetic acid addition in the mobile phase for the present invention.
Fig. 3 changes the chromatogram of first alcohol and water ratio in the mobile phase for the present invention.
The specific embodiment
1, instrument and reagent
Instrument: the ELIIE-P200II type high performance liquid chromatograph that Dalian Yi Lite scientific instrument company produces comprises variable wavelength UV-detector, chromatographic work station, 10 μ L quantitative sample injection rings.
Medicine: the SHENSHUAINING capsule that Lixiang Pharmaceutical Ind. Co. Ltd. produces, lot number 20090814,20090921,20090927;
Reagent: the danshensu sodium reference substance that Chinese medicine and institute of biological products produce, methanol is chromatographically pure, and water is pure water, and other reagent is analytical pure.
2, assay
2.1, chromatographic condition
Chromatographic column: the Inertsil ODS-3 (4.6X150mm 5 μ m) of Dalian Yi Lite scientific instrument company filling;
Mobile phase: methanol: water: the volume proportion of glacial acetic acid is 19: 80: 1;
Detect wavelength: 281nm;
Flow velocity: 1.0ml/min;
Column temperature: 40 ℃;
Theoretical cam curve: be not less than 5000 by the danshensu chromatographic peak.
Chromatographic isolation under these conditions the results are shown in Figure 1, B danshensu peak among the figure.
As shown in Figure 1, component obtains good separation.
2.2, the preparation of reference substance solution
Precision takes by weighing danshensu sodium reference substance 1.66mg, puts in the 10mL measuring bottle, and ultrasonic 10min in 40 ℃ of water-baths takes out, and puts in the ice-water bath 5 minutes, takes out, and add methanol to scale and shake up, as danshensu sodium reference substance liquid, fresh preparation before using.
2.3, the preparation of need testing solution
Accurately take by weighing 12 of SHENSHUAINING capsules (0.35g/ grain) and in the 25mL measuring bottle, crush gently, add methanol 15mL, ultrasonic 10min in 40 ℃ of water-baths, put in the ice-water bath 5 minutes, and took out, add methanol to scale, centrifugal 5min gets supernatant, is SHENSHUAINING capsule test sample liquid.
3, method is investigated
3.1 the selection of condition determination
The influential factor that develops the color is had: bath temperature, water-bath time, concentration.Determine level separately according to documents and materials, each influence factor has been done investigation.
3.2 the investigation of bath temperature
Draw reference substance solution 1.2ml in triangular flask, add 99% methanol solution 1ml immediately, mixing, insulation is 20 minutes in the different temperatures water-bath, takes out, put in the ice-water bath 5 minutes, taking out, is blank with the corresponding reagent, measures absorbance at 281nm wavelength place, the result shows that 40 ℃ of absorbances that record are higher, is bath temperature so select 40 ℃.
3.3, the investigation of methanol concentration
Draw reference substance solution 1.2ml in triangular flask, accurate respectively 85%, 90%, 95%, 99% methanol 1ml, the mixing of adding, insulation is 20 minutes in 40 ℃ of water-baths, takes out, and puts in the ice-water bath 5 minutes, take out, with the corresponding reagent is blank, measures absorbance at 281nm wavelength place, and the result shows, the absorbance that 95% and 99% methanol solution records is better, but 95% dissolve with methanol is inhomogeneous, and sampling amount is inaccurate, is 99% so select methanol concentration.
3.4, the extraction time investigation
Get six parts every part each 1g of SHENSHUAINING medicine 80-100 order coarse powder, the accurate title, decide, and puts in the round-bottomed flask, adds water 100ml, weigh, and reflux 1.5,2.0,2.5 hours, cooling was weighed, and filtered with absorbent cotton, got subsequent filtrate 50ml.Precision is measured subsequent filtrate 2ml, adds ethanol 10ml, shakes up, left standstill 12 hours, centrifugal, get supernatant, put in the 50ml measuring bottle, precision is measured supernatant 2ml in triangular flask, add 99% methanol solution 1ml immediately, mixing, insulation is 20 minutes in 40 ℃ of water-baths, take out, putting in the ice-water bath 5 minutes, and took out, is blank with the corresponding reagent, measure absorbance at 281nm wavelength place, the result shows, reflux 2.0 hours and 2.5 hours are than the content height that refluxed 1.5 hours, and the content of reflux 2.0 hours and 2.5 hours is more or less the same, consider that from the angle of economical rationality choosing return time is 2.0 hours.
3.5, measure the selection of wavelength
Press the preparation method of test sample, preparation danshensu sodium reference substance liquid and SHENSHUAINING capsule test sample liquid, and sample and reference substance liquid carried out spectral scan at the 200nm-400nm place, results sample liquid and reference substance liquid all have absorption maximum at the 281nm place, see Fig. 2, so this assay selects 281nm as measuring wavelength.
3.6, repeatability
Draw SHENSHUAINING capsule test sample liquid, repeat sample introduction 5 times with 20 μ L quantitative sample injection rings, the record peak area, RSD is 0.35%.Get same batch of SHENSHUAINING preparation, by 5 parts of " 2.3 " preparation SHENSHUAINING capsule test sample liquid, with 20 μ L quantitative sample injection ring sample introductions, with external standard method content, the RSD of danshensu measurement result is 0.58%.
3.6, stability
Get same batch of SHENSHUAINING preparation, press the said determination method, every interval 0.5h measures 1 time, records the result and shows, the basically identical as a result that SHENSHUAINING capsule test sample liquid records in 12h.
3.7, the range of linearity
Precision takes by weighing danshensu sodium reference substance 16.55mg, puts in the 50mL measuring bottle, adds methanol constant volume to scale, shakes up.Therefrom precision is measured 1.0mL, 3.0mL, 5.0mL, 7.0mL, 9.0mL respectively, after being diluted to 10mL respectively, respectively get 20 μ L sample introductions, with the chromatographic peak area is vertical coordinate, concentration with danshensu in the solution is abscissa, the drawing standard curve, regression equation is: Y=9320.69X-891.63, r=0.9992.The result shows that danshensu is the good linear relation with peak area value in 0.0298～0.2681mg/mL scope.
3.8, average recovery test
In 5 10mL volumetric flasks, the respectively accurate SHENSHUAINING capsule test sample liquid 4.0mL that adds known content, add 3.0mL, 3.5mL, 4.0mL, 4.5mL, 5.0mL danshensu sodium reference substance liquid more respectively after, be diluted to scale with methanol, shake up.Press method mensuration content under the sample determination item, calculate recovery rate.The average recovery rate of danshensu is 101.28%, and RSD is 0.471%.
3.9, sample determination
Get the SHENSHUAINING capsule sample of 3 lot numbers, get 4 parts for every batch, by " a 2.3 " system SHENSHUAINING capsule test sample liquid.Get test liquid and danshensu sodium reference substance liquid 10 μ L sample introductions respectively, calculate content with peak area by external standard method, measurement result sees Table 1.
The capsular assay result of table 1 SHENSHUAINING (n=3)
| Lot number | ??20090814 | ??20090921 | ??20090927 |
| Average content (mg/g) | ??12.199 | ??11.546 | ??12.381 |
4, conclusion
4.1 in the capsular preparation process of SHENSHUAINING, the extraction of red rooted salvia is mainly based on water extract, therefore, the present invention with the content of water soluble ingredient danshensu in the SHENSHUAINING capsule in the Radix Salviae Miltiorrhizae as quality control standard.Pass through to use the content assaying method of danshensu in the compound recipe SHENSHUAINING capsule in the experiment, its measurement result accuracy height, controllability is strong.Be a kind of can to SHENSHUAINING capsule product quality carry out overall monitor, can be more fast, efficient, and to the less assay method of instrument injury.
4.2 the selection of mobile phase
4.2.1 the amount that glacial acetic acid adds
Under the prerequisite that keeps first alcohol and water constant rate, change the addition of glacial acetic acid in the mobile phase, result such as Fig. 2.
Among Fig. 2: A methanol-water-glacial acetic acid methanol-water-acetic acid (7: 91: 2)
B methanol-water-glacial acetic acid methanol-water-acetic acid (8: 91: 1)
C methanol-water-glacial acetic acid methanol-water-acetic acid (8: 91: 0.1)
In Fig. 2, the T value at danshensu peak is followed successively by 0.99,0.96,0.89.Can find that along with the minimizing of glacial acetic acid addition in the mobile phase, the symmetry at danshensu peak reduces gradually.Because what use in the experiment is the C18 post, along with the increase of glacial acetic acid addition in the mobile phase, the stability of chromatographic column and service life etc. can reduce gradually, and to the maintenance of chromatographic column and also use for a long time unfavorable.Consider the reason of above two aspects, when using peak area to carry out quantitative Analysis, T value between 0.95～1.05 all can, be 1 so select to add in the mobile phase amount of glacial acetic acid.
4.2.2 the ratio of first alcohol and water
Keeping the addition of glacial acetic acid is 1, changes the ratio of first alcohol and water in the mobile phase, result such as Fig. 3.
Among Fig. 3: A methanol-water-glacial acetic acid methanol-water-acetic acid (8: 91: 0.5)
B methanol-water-glacial acetic acid methanol-water-acetic acid (19: 80: 1)
C methanol-water-glacial acetic acid methanol-water-acetic acid (30: 70: 0.1)
The R value at danshensu peak is followed successively by 20.13,9.96 and 1.34, can find.Along with the increase of first alcohol and water ratio in the mobile phase, the separating degree at danshensu peak reduces gradually, and in the sample all component stream go out the chromatographic column required time and increase gradually.Along with the prolongation of minute, chromatographic peak easily drifts about, and required mobile phase increases the cost up of experiment.At least should consider factors such as analysis time and separating degree greater than 1.5 regulation according to separating degree, select for use methanol-water-glacial acetic acid (19: 80: 1) to be mobile phase.
4.3 production extracting method
With SHENSHUAINING capsule Chinese crude drug, be loaded in the polyethylene tube internal diameter 100cm, sample length 150cm.The polyethylene tube two ends clog with absorbent cotton, and an end connects peristaltic pump, and an end connects the sample accepter.This polyethylene tube is put into microwave cavity, make raw material be positioned at 3/4 wavelength place of this resonator cavity.Extractant enters by peristaltic pump, enters receptor behind the material of flowing through, and receives the 3000kg extract altogether.
The relative retention time of total fingerprint peaks in the table 2 SHENSHUAINING capsule collection of illustrative plates
Total fingerprint peaks relative peak area in the table 3 SHENSHUAINING capsule fingerprint pattern
Finger printing similarity among the table 4 SHENSHUAINING capsule HPLC
| Sample number into spectrum | ??S1 | ??S2 | ??S3 | ??S4 | ??S5 | ??S6 | ??S7 | ??S8 | ??S9 | ??S10 | ??S11 | ??S12 | ??S13 | Reference fingerprint |
| Similarity | ??0.986 | ??0.984 | ??0.979 | ??0.917 | ??0.905 | ??0.913 | ??0.969 | ??0.948 | ??0.973 | ??0.987 | ??0.988 | ??0.962 | ??0.943 | ??1.000 |
Claims (3)
1. use the HPLC method to measure the method for content of Danshensu in the SHENSHUAINING capsule, it is characterized in that step is as follows:
(1) SHENSHUAINING capsule test solution preparation: accurately take by weighing 12 of SHENSHUAINING capsules (0.35g/ grain) in the 25mL measuring bottle, crush gently, add methanol 15mL, ultrasonic 10min in 40 ℃ of water-baths, put in the ice-water bath 5 minutes, and took out, add methanol to scale, centrifugal 5min gets supernatant and is the Yiganning capsule test solution that declines;
(2) preparation of reference substance solution: precision takes by weighing danshensu sodium reference substance 1.66mg, puts in the 10mL measuring bottle, and ultrasonic 10min in 40 ℃ of water-baths takes out, and puts in the ice-water bath 5 minutes, takes out, and adds methanol to scale and shakes up, and is reference substance solution;
(3) chromatographic condition: chromatographic column is Inertsil ODS-3 (4.6X150mm 5 μ m); Mobile phase is methanol-water-glacial acetic acid, and volume ratio is 19: 80: 1; Flow velocity: 1.0ml/min; The detection wavelength is 281nm; Sensitivity: 0.01AUFS; Sample size: 10 μ L; Column temperature: 40 ℃, theoretical cam curve: be not less than 5000 by the danshensu chromatographic peak;
(4) algoscopy: accurate respectively danshensu sodium reference substance solution and each 10mL of SHENSHUAINING capsule test solution of drawing, inject chromatograph of liquid, measure SHENSHUAINING capsule test solution and should present and the identical chromatographic peak of danshensu sodium reference substance solution chromatographic retention.
2. use HPLC method according to claim 1 is measured the method for content of Danshensu in the SHENSHUAINING capsule, it is characterized in that, existing preparation before the described danshensu sodium reference substance of step (2) uses, lucifuge, cryopreservation.
3. use HPLC method according to claim 1 is measured the method for content of Danshensu in the SHENSHUAINING capsule, it is characterized in that the methanol that is adopted is chromatographically pure, and water is pure water, and other reagent is analytical pure.
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| CN101816715B (en) | 2012-03-28 |
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Denomination of invention: Method for measuring content of Danshensu in shenshuaining capsules by utilizing HPLC method Effective date of registration: 20141117 Granted publication date: 20120328 Pledgee: China Merchants Bank Limited by Share Ltd. Kunming hi tech sub branch Pledgor: Luo Shi|YUNNAN LIXIANG PHARMACEUTICAL Co.,Ltd. Registration number: 2014990000950 |
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